Protective effect of Enicostema axillare on mutagenicity of Salmonella typhimurium by increasing free radical scavenging abilities

Figures & data

Table 1.  Effect of fractions of E. axillare to S. typhimurium spontaneous revertants in the presence (+S9) or absence of (−S9) microsomal fraction.

Fractions (3 mg/plate)	Average no. of colonies revertants/plate
	TA98		TA100
	S9 (+)	S9 (-)	S9 (+)	S9 (-)
Hexane	47 ± 0.95^a	41 ± 1.32^a	71 ± 1.11^a	63 ± 2.13^a
Ethyl acetate	51 ± 2.43^a	49 ± 1.12^a	69 ± 0.54^a	61 ± 2.22^a
Methanol	49 ± 1.23^a	44 ± 1.64^a	69 ± 2.32^a	59 ± 1.68^a
Spontaneous revertants	51 ± 1.74^a	42 ± 1.49^a	72 ± 1.43^a	61 ± 1.91^a

Values represent as mean ± SEM for triplicate.

Values carrying same alphabet did not vary significantly from each other (Tukey’s HSD; P ≤ 0.05).

Table 2.  Effects of fractions of E. axillare on mutagenesis of NaN₃, NBT and 2-AF in TA98 strain of S. typhimurium.

Groups	NaN3 (0.0025 mg/plate)		NBT (0.02 mg/plate)		2-AF (0.02 mg/plate)
	Pre-incubation	Co-incubation	Pre-incubation	Co-incubation	Pre-incubation	Co-incubation
Positive control	1120.00 ± 5.50^a		1340.00 ± 9.01^a		980.00 ± 7.50^a
Hexane (1 mg/plate)	680.00 ± 5.50^b	763.33 ± 3.71^b	960.00 ± 4.16^b	1180.00 ± 4.58^b	642.00 ± 3.05^b	740.00 ± 5.50^b
Ethyl acetate (1 mg/plate)	544.00 ± 1.73^d	620.00 ± 6.65^d	660.00 ± 4.93^d	730.66 ± 0.33^e	448.00 ± 4.61^d	456.00 ± 3.78^d
Methanol (1 mg/plate)	600.00 ± 6.50^c	678.66 ± 7.51^c	700.00 ± 8.02^c	824.00 ± 3.05^c	540.00 ± 4.50^c	568.00 ± 3.05^c
Hexane (2 mg/plate)	320.00 ± 2.51^e	440.00 ± 7.50^e	695.33 ± 1.76^c	766.00 ± 3.05^d	321.00 ± 5.50^f	360.00 ± 7.02^f
Ethyl acetate (2 mg/plate)	288.00 ± 4.35^f	328.00 ± 4.16^h	440.00 ± 6.11^g	520.00 ± 5.50^g	220.00 ± 3.05^g	250.00 ± 2.51^g
Methanol (2 mg/plate)	324.00 ± 2.51^e	496.00 ± 6.11^g	520.00 ± 2.64^e	640.00 ± 3.05^f	382.00 ± 1.00^e	430.00 ± 3.05^e
Hexane (3 mg/plate)	280.00 ± 3.78^f	390.00 ± 7.37^f	479.33 ± 8.81^f	520.00 ± 9.07^g	240.00 ± 7.76^g	280.00 ± 3.05^f
Ethyl acetate (3 mg/plate)	170.00 ± 4.58^h	209.66 ± 1.76^i	280.00 ± 3.21^i	280.00 ± 2.08^i	140.00 ± 8.02^h	170.00 ± 4.04^h
Methanol (3 mg/plate)	254.00 ± 2.51^g	410.00 ± 2.51^f	380.00 ± 2.51^h	466.00 ± 3.00^h	220.00 ± 3.05^g	260.00 ± 3.05^g

Values represent as mean ± SEM for triplicate.

Values carrying same alphabet did not vary significantly from each other (Tukey’s HSD; P ≤ 0.05).

Table 3.  Effect of fractions of E. axillare on mutagenesis of NaN₃, NBT and 2-AF in TA100 strain of S. typhimurium.

Groups	NaN3 (0.0025 mg/plate)		NBT (0.02 mg/plate)		2-AF (0.02 mg/plate)
	Pre-incubation	Co-incubation	Pre-incubation	Co-incubation	Pre-incubation	Co-incubation
Positive control	1186.00 ± 5.50^a		1493.00 ± 7.63^a		1009.00 ± 5.85^a
Hexane (1 mg/plate)	664.00 ± 1.52^b	753.00 ± 3.05^b	993.00 ± 3.78^b	1043.00 ± 10.40^b	642.00 ± 4.72^b	704.00 ± 2.00^b
Ethyl acetate (1 mg/plate)	514.00 ± 3.51^d	602.00 ± 2.51^d	679.00 ± 3.21^d	723.00 ± 2.51^d	409.00 ± 4.50^d	532.00 ± 3.51^d
Methanol (1 mg/plate)	600.00 ± 5.50^c	672.00 ± 4.16^c	713.00 ± 2.51^c	782.00 ± 3.51^c	501.00 ± 3.46^c	573.00 ± 2.08^c
Hexane (2 mg/plate)	410.00 ± 3.21^e	502.00 ± 4.58^e	724.00 ± 6.08^c	794.00 ± 3.05^c	337.00 ± 4.35^e	411.00 ± 5.03^e
Ethyl acetate (2 mg/plate)	253.00 ± 0.577^h	328.00 ± 0.57^g	463.00 ± 4.50^f	551.00 ± 0.57^f	196.00 ± 2.51^h	272.00 ± 4.58^g
Methanol (2 mg/plate)	324.00 ± 4.50^f	496.00 ± 2.51^e	513.00 ± 2.51^e	617.00 ± 2.51^e	298.00 ± 0.57^f	401.00 ± 3.78^f
Hexane (3 mg/plate)	292.00 ± 3.05^g	401.00 ± 3.05^f	493.00 ± 1.15^e	563.00 ± 3.05^f	234.66 ± 3.33^g	261.00 ± 5.50^g
Ethyl acetate (3 mg/plate)	186.00 ± 1.15^i	232.00 ± 3.46^i	284.00 ± 3.60^g	342.00 ± 3.00^g	152.66 ± 4.70^i	188.00 ± 1.00^h
Methanol (3 mg/plate)	237.00 ± 3.78^h	272.00 ± 3.51^h	331.00 ± 1.15^h	386.00 ± 4.04^h	214.00 ± 2.08^h	275.00 ± 3.05^g

Values represent as mean ± SEM for triplicate.

Values carrying same alphabet did not vary significantly from each other (Tukey’s HSD; P ≤ 0.05).

Figure 1.  Percentage inhibition of mutagenesis by the preincubation and coincubation modes of experiments with E. axillare fractions against NaN3 induced mutagenicity on TA98 (A, preincubation and B, coincubation) and TA100 (C, preincubation and D, coincubation). ([GRAPHIC], hexane extract; [GRAPHIC], ethyl acetate extract; [GRAPHIC], methanol extract). Values carrying different alphabets vary significantly from each other (Tukey’s HSD; p ≤ 0.05).

Figure 2.  Percentage inhibition of mutagenesis by the preincubation and coincubation mode of experiments with E. axillare fractions against NPD-induced mutagenicity on TA98 (A, preincubation and B, coincubation) and TA100 (C, preincubation and D, coincubation). ([GRAPHIC], hexane extract; [GRAPHIC], ethyl acetate extract; [GRAPHIC] methanol extract). Values carrying different alphabets vary significantly from each other (Tukey’s HSD; p ≤ 0.05).

Figure 3.  Percentage inhibition of mutagenesis by the preincubation and coincubation mode of experiments with E. axillare fractions against 2-AF induced mutagenicity on TA98 (A, preincubation; B, coincubation) and TA100 (C, preincubation; D, coincubation). ([GRAPHIC], hexane extract; [GRAPHIC], ethyl acetate extract; [GRAPHIC], methanol extract). Values carrying different alphabets vary significantly from each other (Tukey’s HSD; p ≤ 0.05).

Table 4.  Phytochemical analysis and antioxidant capacity of the fractions from E. axillare.

Fractions	Polyphenol content of the fractions (mg gallic acid equivalent/g fraction)	Total flavonoid content of the fractions (mg rutin equivalent/g fraction)	DPPH scavenging effect of the fractions (IC50 values in µg)	Total antioxidant capacity of the fractions (Ascorbic acid equivalents µg AA/100 µg fraction)
Hexane	70.33 ± 2.72^a	0.41 ± 0.25^a	> 1000	07.35 ± 0.57^a
Ethyl acetate	263.67 ± 6.70^b	38.48 ± 1.85^b	192.27 ± 3.67; r^2 − 0.756^b	24.79 ± 0.29^b
Methanol	283.65 ± 3.94^c	44.49 ± 1.24^c	188.77 ± 3.67; r^2 − 0.658^c	17.32 ± 0.57^c

Values represent as mean ± SEM for triplicate.

Values carrying same alphabet did not vary significantly (Tukey’s HSD; P ≤ 0.05).

Table 5.  Effect of E. axillare fractions on various serum biochemical parameters.

Parameters	Normal control	Hexane (200 mg/kg bw)	Ethyl acetate (200 mg/kg bw)	Methanol (200 mg/kg bw)
ALP (U/l)	92.65 ± 0.77^a	91.54 ± 0.51^a	92.01 ± 0.80^a	92.03 ± 0.46^a
AST (U/l)	55.32 ± 0.81^a	57.23 ± 0.80^a	56.77 ± 0.59^a	57.31 ± 0.67^a
ALT (U/l)	18.54 ± 0.81^a	16.74 ± 0.58^a	16.04 ± 1.31^a	16.26 ± 0.89^a
LDH (U/l)	494.96 ± 3.32^a	489.64 ± 2.60^a	493.65 ± 2.65^a	492.75 ± 0.85^a
Creatinine (µmol/l)	36.79 ± 0.98^a	37.02 ± 0.78^a	37.02 ± 0.78^a	36.85 ± 0.62^a
Total protein (g/l)	36.87 ± 0.62^a	36.04 ± 0.43^a	34.87 ± 1.32^a	35.99 ± 1.17^a

Values represent as mean ± SEM for triplicate.

Values carrying same alphabet did not vary significantly (Tukey’s HSD; P ≤ 0.05).

Figure 4.  Histopathology of liver tissue from mice exposed to the fractions of E. axillare at a dose of 200 mg/kg bw after 21 days postoral administration. (A) Control animals, (B) hexane fraction of E. axillare, (C) ethyl acetate fraction of E. axillare, (D) methanol fraction of E. axillare.